From physiology to biochemistry to spectroscopy

and protein structure: copper and iron proteins Activation and Transformation of Dioxygen by Iron and Copper Enzymes

Otto H. Warburg

Nature Reviews (2011)

11, 325

Otto Heinrich Warburg (1883–1970), was a German physiologist,

medical doctor and Nobel laureate. He earned double doctorates in

chemistry and medicine, and won the Nobel Prize in 1931, for his

research into cellular respiration, showing that cancer thrives in

anaerobic (without oxygen) or acidic conditions. His father was a

highly respected physicist, and in Warburg's childhood such

luminaries as Albert Einstein, Max Planck, Emil Fischer, and

Walther Nernst were frequent dinner guests. OW was one of the

twentieth century's leading biochemists. He was nominated an

unprecedented three times for the Nobel prize for three separate

achievements.

"Cancer, above all other diseases, has countless secondary causes.

But, even for cancer, there is only one prime cause. Summarized in

a few words, the prime cause of cancer is the replacement of the

respiration of oxygen in normal body cells by a fermentation of

sugar.” O. Warburg (1956), "On the origin of cancer cells", Science 123, 309–14

1

Metal-O2 Inorganic Biological Chemistry

B. G. Malmstroem (1982) Enzymology of Oxygen. Ann. Rev. Biochem., 51, 21-59

H. Beinert (1995) Crystals and structures of cytochrome c oxidases – the end of an arduous

road. Chem. Biol., 2, 781-785

B. G. Malmstroem (1997) A life with the metals of life. Selected Topics in the History of

Biochemistry Comprehensive Biochemistry, 40, 277-331

M. Saraste (1999) Oxidative Phosphorylation at the fin de siècle. SCIENCE, 283, 1488-

1493

I. Schlichting, J. Berendzen, K. Chu, A. M. Stock, S.A. Maves, D. E. Benson, R. M.

Sweet, D. Ringe, G.A. Petzko, S.G. Sligar (2000) The Catalytic Pathway of Cytochrome

P450cam at Atomic Resolution, SCIENCE, 287, 1615-1622

J. A. Kovacs (2003) How Iron Activates O2. SCIENCE, 299, 1024-1025

W.B. Tolman (2006) Using synthetic chemistry to understand copper protein active sites: a

personal perspective. J. Biol. Inorg. Chem., 11, 261-271

L. Que Jr, W.B. Tolman (2008) Biologically inspired oxidation catalysis. NATURE, 455,

333-340 2

Dioxygen Activation

ss

ss*

sp*

sp

p*

p

3S+

O2

O2•-

H2O2

•OH+H2O

2H2O

-0.33 V

+0.94 V

+0.38 V

+2.31 V

E0 vs. NHE at pH 7.25

112 pm

133 pm

1.49 pm

1554 cm-1

1145 cm-1

842 cm-1

3

Activation of O2 – Reaction Types

• Reversible binding of O2 – Myoglobin, Hemoglobin (Fe), Hemocyanin

(Cu-Cu)

• O2.- dismutation – Superoxide Dismutase (Mn, Fe, Ni, Cu, Zn)

O2.- + O2

.- +2H+ → O2 + H2O2

• H2O2 decomposition – Catalase (Mn, heme-Fe)

2 H2O2 → 2 H2O + O2

• Oxygenases (focus on Monooxgenase Cytochrome P450)

R-H + O2 + NADPH + H+ → R-OH + H2O + NADP+

• Oxidases (2-electron reduction to H2O2; Fe, Cu)

O2 + 2e- +2H+ → H2O2 (focus on Cu enzyme Galactose Oxidase)

• Oxidases (4-electron reduction to H2O; heme-Fe, Cu)

O2 + 4e- +4H+ → 2 H2O (focus on Cu enzyme Ascorbic Acid Oxidase

and Fe,Cu enzyme Cytochrome c Oxidase)

4

O2 activation by Metallo-Oxygenases Mechanisms involve the formation of an initial O2 adduct (superoxo), conversion to a

metal–peroxide (peroxo), and subsequent O–O bond cleavage to yield a high-valent

oxidant (oxo). Oxygen atoms involved are shown in red. M, metal; P, porphyrin. L. Que Jr, W.B. Tolman (2008) NATURE, 455, 333-340

5

Reversible O2 Binding

Myoglobin and Hemoglobin

The iron must be in the Fe(II) (ferrous

oxidation) state. 6

Binding of O2 rearranges the

electronic distribution and

alters the d orbital energy.

This causes a difference in the

absorption spectra.

Bluish for deoxy Hb, Redish

for Oxy Hb

Measuring the absorption at

578 nm allows an easy method

to determine the percent of O2

bound to hemoglobin.

7

Hemocyanin (reversible O2 binding)

8 K Magnus, Limulus polyphemus (atlantic horseshoe crab) Hemocyanin, PDB 1OXY,

Handbook of Metalloproteins (2001)

Oxygenated Cu site, see Que, Tolman,

NATURE (2008) 455, 333; oxygenated form

has a blue colour, μ-η2:η2-peroxo binding

mode

Cu,Zn Superoxide Dismutase (SOD) PDB code 1SPD http://en.wikipedia.org/wiki/Superoxide_dismutase

9

M(n+1)+-SOD + O2− → Mn+-SOD + O2

Mn+-SOD + O2− + 2H+ → M(n+1)+-SOD + H2O2

HUMAN ERYTHROCYTE CATALASE

PDB 1DGB C.D. Putnam, A.S. Arvai, Y. Bourne, J.A Tainer, J. Mol. Biol. (2000) 296, 295–309

10

H2O2 + Fe(III)-E → H2O + O=Fe(IV)-E(.+)

H2O2 + O=Fe(IV)-E(.+) → H2O + Fe(III)-E + O2

Porphyrin Radical Cation

O=Fe(IV)-E(.+) is involved in the

reaction mechanism

http://en.wikipedia.org/wiki/Catalase

Tyrosine acts as axial ligand Maté et al. (2001) (Messerschmidt, Huber, Poulos, Wieghardt (eds)) Handbook of Metalloproteins, John

Wiley & Sons, LTD

11

Fe(IV)=O state

Cytochrome P450

12 Ortiz de Montellano Chem. Rev. (2010) 110, 932–948; Denisov,

Makris, Sligar,Schlichting, Chem. Rev. (2005) 105, 2253-2277

Trans-Effect - Tuning reactivity

A ligand X trans to a second ligand Y can influence the stability of the M-Y

bond. With X being a strong Lewis base, the M-Y bond will be weakened

RH + O2 + 2H+ + 2e- → R-OH + H2O

13

Typical Reactions of Cytochrome P450 (O-Transfer)

S. SHAIK et al. (2010) Acc. Chem. Res., 43, 1154-1165

J. Rittle, M. T. Green (2010), SCIENCE 330, 933-936

14

Cytochrome P450 – important enzyme for

detoxification of organic compounds

15

Camphor substrate complex of

Cytochrome P450

16

Cytochrome P450 Reaction Cycle

compound I

key intermediate

Fe(II)OH2

Fe(III)O2-

Fe(III)-OOH

Fe(V)=O

Fe(III)OH

H2O

17

Galactose Oxidase, a Cu-Ligand Radical Enzyme

RCH2OH + O2 RCHO + H2O2 GalOx

2 different EPR signals (metal-centered

and ligand centered) observed during reaction cycle

ABS

18

Galactose Oxidase - Mechanism Que, Tolman (2008) NATURE 455, 333

19

Galactose Oxidase - Mechanism Que, Tolman (2008) NATURE 455, 333

20

History: The discovery of a new Copper Center D KEILIN, T MANN, Nature, 143, 23-24 (1939)

BG MALMSTRÖM, R MOSBACH, T VÄNNGÅRD, Nature, 183, 321-322 (1959)

1939: Laccase, a Blue Copper-Protein Oxidase from the Latex of Rhus

succedanea

1959: An Electron Spin Resonance Study of the State of Copper in Fungal

Laccase (a blue Multi-Copper oxidase)

Type 1 Blue Copper Electron Transfer Center E I Solomon, Inorg. Chem., 45, 8012-8025 (2006)

21

Plastocyanin: Blue Type 1 Cu Site

Function: Electron Transfer Protein/Photosynthesis

41% Cu 38-45% S

Cu(II) Spin-Distribution

Covalent Cu-Cys p-bond is mainly responsible

for its unique properties

EI Solomon, Inorg. Chem. 2006, 45, 8012-8025

PDB Code: 1PLC

HC Freeman, 1978 22

Ascorbic acid oxidase (AOX) Multi-Copper oxidase (8Cu/homodimer) PDB code 1AOZ

23

(5R)-[(1S)-1,2-dihydroxyethyl]-3,4-

dihydroxyfuran-2(5H)-one

Type 1 Cu ET center in AOX A. Messerschmidt, Handbook of Metalloproteins (2001)

24

Entrance point for electrons

ß-barrel structural module

25

Trinuclear Cu center in AOX

Dioxygen reduction site

Reduced Trinuclear Site and Peroxide Adduct of AOX

26

ET Pathways in AOX

27

Longe Range Electron Transfer through Protein Early experiments by H Gray and J Winkler/Caltech

Ru-Complex

covalently attached

to Fe protein

cytochrome c

RA Marcus Nobel Prize

Chemistry 1992

28

Electron transfer: Marcus Theory

Nuclear coordinates

Energ

y

ABH2

Tk

B

AB

ETBe

Tk

Hk

p 4

2

2 2

optE

actE

oxredredox BABA

Factor"Electronic"

2

Factor"CondonFranck"

42/12

20

44

AB

kT

G

ET HekTh

k

pp

=Reorganization energy

( structural change upon ET)

0G = Driving force

( difference of potentials donor vs

acceptor)

ABH = Electronic Coupling

(“overlap of orbitals”)

29

Low (zero) Reorganization Energy

Sigfridsson, E.; Olsson, M.H.M.; Ryde, U. (2001) J. Phys. Chem. B, 105, 5546

FeIII

NHis

NHis

FeII

NHis

NHis

199.4 pm 200.9 pm

e-

Reorganization Energy

in Cytochromes 4-5 kcal/mol

Low-Spin Heme center

30

Opening of Substrate Binding Sites in Enzyme

Davis, M.I.; Orville, A.M.; Neese, F.; Zaleski, J.; Lipscomb, J.D.; Solomon, E.I. (2002) J. Am. Chem. Soc. 124, 602

+Substrate

- Tyrosine

3,4 Protocatechuate Dioxygenase

31

Cytochrome c oxidase (COX), a redox-driven proton pump Proton-Coupled Electron Transfer in COX

Kaila, Verkhovsky, Wikstroem, Chemical Reviews (2010) 110, 7062–7081

32

Cytochrome c oxidase S. Yoshikawa, K. Muramoto, K. Shinzawa-Itoh Annu. Rev. Biophys. (2011) 40, 205–23

Tomoya Hino, et al. SCIENCE (2010) 330, 1666-1670

O2 + 4H+ + 4Hi+ + 4e- H2O + H2O + 4Ho

+ (+ 818 mV) N2O + 2H+ 2e- N2 + H2O (+1355 mV) 2NO + 2H+ + 2e- N2O + H2O (+1175 mV)

metals (CuA, Fe-heme, Mg, Zn) e- transfer (redox; tyrosyl radical ?), H+transfer (pump) metal centers: CuA ET; Fe-CuB O2 reduction

33

Cytochrome/Heme Types

Heme a Heme b Heme c

34

Cytochrome c oxidase

M Saraste Science (1999) 283,1488-1493

Published by AAAS

Mitochondrial Cytochrome c oxidase (COX) (representation of the monomer from bovine heart/13 subunits)

Tsukihara et al., SCIENCE 1995, 269, 1069; Yoshikawa et al., SCIENCE 1998, 280, 1723

Bacterial COX from Pseudomonas denitrificans (three subunits; Iwata et al., NATURE, 1995, 376, 660)

36

Metal Centers in bacterial COX

Cu Fe Mg

37

NO Reductase (NOR) T. Hino, et al. SCIENCE (2010) 330, 1666-1670

38

Site of O2 Reduction (Fe(III)-Cu(II) State – covalent link Tyr-His)

39

Active Sites of NOR (A) and COX (B)

40

Schematic Representation of the Catalytic Cycle

(no release of toxic ROS; tyrosine radical Y)

41

Proton Transfer Pathways

(D Asp 124 & E Glu 78) in P. denitrificans COX

42

Aspartate 51 (D51/51) – Site of Proton Pumping ? (observation of a redox induced conformational change)

43

Sir Humphry

Davy (1800) Researches, chemical

and philosophical

–chiefly concerning

nitrous oxide or

dephlogisticated

nitrous air, and its

respiration.

What Is Nitrous Oxide?

A former nitrous addict and expert on addiction

explains why laughing gas is no joke….

By Maia Szalavitz | @maiasz | January 26, 2012.

Actress D… M….’s trip to the emergency room was

reportedly spurred by symptoms of a seizure

triggered by inhaling the drug nitrous oxide, more

commonly known as laughing gas or whippits.

N2O has many

uses….

44

N N O N N O

45

Nitrous Oxide – Dinitrogen Monoxide

Laughing Gas – Sweet Air

A Structurally Characterized Nitrous

Oxide Complex of Vanadium

Piro et al., J. Am. Chem. Soc. (2011) 133,

2108–2111

N2O – A Potent Greenhouse Gas Wuebbles, Science (2009), 326, 56-57

N2O → NOx → Ozone effects

Stratosphere

N2O + IR → Climate effects

Troposphere

46 http://epa.gov/climatechange/emissions

/usinventoryreport.html

N2O + 2H+ + 2e- N2 + H2O E’o = +1.35 V

kinetically inert molecule

~ 59 kcal/mol activation barrier

47

Bacterial Nitrous Oxide Reductase is a

purple Copper Enzyme

Bacterial Nitrous Oxide Reductase is a head-to-tail homodimer,

with 6 Cu/monomer A. Pomowski, W.G. Zumft, P.M.H. Kroneck, O. Einsle (2011) Nature 477, 234-237

48

.

There are two novel Copper-Sulfur sites

(A) the dinuclear CuA, (B) the tetranuclear CuZ

49

• Crystals grown in N2/H2

atmosphere, flash-frozen

in liquid nitrogen

• N2OR crystals are stable, high

diffraction quality

Data collection at Paul Scherrer

Institute, Swiss Light Source,

Villigen (CH)

Protein Crystallography and in crystallo Chemistry

50

O Einsle

In Bio EPR nothing compares to the returns

from a standard X-band instrument (9-10 GHz)

2600 2800 3000 3200 3400

Magnetic Field (G)

7-Line spectrum

X-band, 15 K

Coyle CL, Zumft WG, Kroneck PMH, Körner H, Jakob W (1985)

Purple Nitrous Oxide Reductase. Eur. J. Biochem., 153, 459-467 51

W Zumft

Microbiologist

CuA is Mixed-Valence Cu2S2 Rhomb, S=1/2 formal oxidation state of Cu 1.5+, 1 unpaired electron/2Cu

52

F Neese

Cu-Cu Bond ?

(Metallic Cu)

CuA is also present in respiratory Cytochrome c oxidase Neese F, Zumft WG, Antholine WE, Kroneck PMH (1996) J. Am. Chem. Soc., 118, 8692-8699

Cu Fe Mg

O2 reduction site

O2 → H2O

Electron entrance

Pathways for

electrons & protons

53

Evolution and Bioengineering through Loop directed Mutagenesis

From a Blue Mononuclear Cu to a Purple Dinuclear CuA MG Savelieff, Y Lu, J Biol Inorg Chem, 15, 967-976 (2010)

Plastocyanin/Photosynthesis Nitrous Oxide Reductase/Denitrification

Cytochrome c Oxidase/Respiration 54

CuZ is a Copper Sulfide (S2-) complex which is oxygen-sensitive First Xtal structures showed oxygen-ligands coordinated instead of sulfide

CuZ was proposed to activate N2O upon binding at position X

SI Gorelsky, S Ghosh, EI Solomon (2006) J. Am. Chem. Soc. 128, 278-290

X

S

Cu

Cu

Cu Cu

NHis

NHisNHis

NHis

NHis

NHis

NHis

S=0

S=1/2

Cu4IIS2-

6+

Cu3IICu1

IS2-5+

Cu2IICu2

IS2-4+

Cu1IICu3

IS2-3+

Cu4IS2-

2+

+e-

+e-

+e-

+e-

-e-

-e-

-e-

-e-

55

When prepared/crystallized under the strict

exclusion of O2, CuZ is a Cu4S2 Cluster

56

Upon binding of N2O (Xtal under N2O pressure) the ligands of

CuA rearrange and His 583 flips to ligate Cu1 of CuA

57

Catalysis: CuA and CuZ operate in concert Xtal under N2O pressure

58

Take Home Message

59

“Dear Lord, I fall upon my knees

and pray that all my syntheses

may cease to be inferior

to those conducted by bacteria”

Leslie D. Pettit, X. International Symposium on

Bioinorganic Chemistry, Szklarska Poreba, Poland

(2005)

Bacteria are outstanding Chemists

To advance in SCIENCE and ARTS,

YOU need a good EDUCATION,

YOU must have IDEAS, even some crazy ones,

however, 95-98% will be HARD WORK...

60